Approximately one-third of the total protein in mammalian organisms is comprised of collagen; the main constituent of skin, tendon, cartilage, teeth and bone material is collagen. Mandl, I., COLLAGENASE, "Collagenase Comes of Age", p. 1 (Gordon and Breach, Science Publishers, Inc., 1972). Enzymes which degrade collagen are of importance in controlling conditions involving collagen rich tissue. Collagenase is effective in controlling such conditions.
The use of enzymes in the medical field is well known. For example, Collagenase produced from the bacterium Clostridium histolyticum has been used as a debridement agent. Other enzymes are used surgically in ophthalmology. For example, alphachymotrysin is used to lyse zonules in cataract surgery and hyaluronidase is used extraocularly as a means of spreading local anesthesia more effectively through tissue.
The use of enzymes and, in particular, collagenase has been disclosed in the art. Processes for the production of Collagenase from Clostridium histolyticum have been disclosed in U.S. Pat. Nos. 3,705,083 and 3,821,364. Processes for preparing other Collagenases have been disclosed in U.S. Pat. Nos. 3,267,006 and 3,677,900. Collagenase has been used in the treatment of herniated intervertebral discs of mammals and such treatment is disclosed in U.S. Pat. No. 3,678,158. Detoxified enzymes obtained from snake venom have been used in the treatment of ocular disorders as disclosed in U.S. Pat. No. 3,869,548.
The medical use of enzymes in the ocular region and, in particular, the medical use of collagenase in the ocular region presents the investigator with a multitude of problems. In general, the mammalian eyeball and surrounding tissue is comprised of a large quantity of collagen. Because of this, any injection or use of collagenase in the ocular region could potentially damage tissue not intended to be attacked by the collagenase.
The following brief discussion concerning basic ocular anatomy and opthalmic surgery serves to describe the many collagen rich structures of the ocular region and the problems facing opthalmic surgeons which are caused by the collagen rich structures.
Quite basically, the mammalian eyeball may be divided into an anterior and posterior portion, each of which has three layers. The outer most layer, the sclera, consists of tough connective tissue. The sclera is continuous with the transparent cornea anteriorly. The middle or vascular layer, properly called the choroid, is continuous anteriorly with the ciliary body and the pigmented portion of the eye, the iris. The retina comprises the internal layer of the eye.
The vitreous body is a transparent gel-like material which occupies approximately 80 percent of the posterior of the eyeball. On the anterior surface of the vitreous and posterior to the iris and pupil is the lens which is a transparent, biconvex and circular structure. The lens consists of many concentric layers of proteinaceous material surrounded by a capsule and held in position by suspensory ligaments.
Between the transparent cornea anteriorly and the iris posteriorly is the anterior chamber which contains aqueous humor. The anterior chamber is directly connected with the small posterior chamber of the eye via the pupillary opening. Aqueous humor is secreted by cells of the ciliary body. This fluid flows into the posterior chamber and through the pupil into the anterior chamber in order to nourish the cornea and maintain internal ocular pressure. In humans, this aqueous fluid is formed at an approximate rate of 2.1 microliters/minute. The volume of the anterior chamber is 0.25 milliliters and this fluid which flows into the chamber is filtered out through a system of channels in the trabecular meshwork.
The sclera, composed essentially of collagen, is a white, opaque covering of the eyeball and is continuous with the cornea. Anteriorly, the sclera is covered by the conjunctiva, a mucus membrane coating.
The cornea is composed of five layers, the corneal epithelium anteriorly, Bowman's membrane, the corneal stroma, Descement's membrane and the endothelium. The corneal epithelium is a continuation of the cells of the conjunctiva. The entire posterior cornea is covered by endothelial cells which function to maintain the cornea's crystalline clarity. Small molecules with a molecular weight of less than 6,200 can penetrate the endothelium membranes and enter the cornea. Molecules of larger molecular weight are unable to penetrate the endothelium. Bowman's membrane, the stroma, the Descement's membrane are all composed of collagen.
The lens is completely surrounded by a collagenous capsule. Beneath the anterior capsule are epithelial cells. The fibers of the lens are composed of hard crystalloid protein which is slowly secreted by the lens cells throughout life.
The iris, located at the anterior portion of the eye, is a thin circular disc which functions in a manner similar to the diaphragm of a camera. The iris consists of four layers: the anterior border layer, the stroma, the dilator muscle, and the posterior epithelium. Blood vessels located in the iris and the stroma contain collagenous tissue.
The trabecular meshwork is a system of filters located in the angle of the anterior chamber. This meshwork consists of a collection of collagenous pillars which are lined by endothelial cells. It is the trabecular meshwork through which the aqueous fluid flows by entering a gradually enlarging system of collector channels to enter the aqueous veins and ultimately leave the anterior portion of the eye.
The vitreous or hyaloid body is a transparent gel which is bound anteriorly by the lens and posteriorly by the internal limiting membrane of the retina. The attachment is strongest at the peripheral retina and the ciliary body. This attachment is known as the vitreous base. Basically, the vitreous consists of a three dimensional structure of straight collagen fibrils in whose interstices are loosely wound molecules of hyaluronic acid. The collagen fibrils are thickest at the peripheral vitreous, especially so at the vitreous base.
The retina consists of several cell layers. The outermost is the rod and cone layer which transforms light impulses into neurotransmitter impulses. Behind this layer lie the association neuronal cells. The innermost layers consist of ganglion cells which transmit information via the optic nerve to the brain. The inner limiting membrane is adjacent and posterior to the vitreous. Retinal blood vessels in the human run in the inner retinal layers and do not protrude into the vitreous. These retinal blood vessels contain collagen. In some animals, for example rabbits, these blood vessels protrude into the vitreous.
To summarize the human's ocular anatomy, a major portion of the protein constituent of the ocular region consists of collagen. In particular, the major protein constituent of the vitreous is collagen. Other ocular structures such as the cornea, and the trabecular meshwork contain collagen and each of these structures is lined by noncollagenous cells. Additionally, retinal blood vessels are comprised of collagenous material; however, these vessels lie within and not on the surface of the human retina.
The fact that many of the structures in the ocular region are composed of collagenous material would not appear to be of any great concern; however, many types of ophthalmic surgery, ocular region injuries, and ocular diseases, are complicated by structure rich in collagen fibrils. Moreover, intraocular scar formation which is a natural physiological response to trauma, including surgical trauma, can seriously affect vision since the intraocular space is so small and the many structures of the eye which are necessary for maintaining visual function are in close proximity. In many cases, glaucoma and retinal detachment are caused by intraocular scar formation. Additionally, scars formed on or under the conjunctiva, subconjunctival area, could damage the cornea and scars on the eyelids could impair proper visual function. Collagen is a major structural constituent of scars.
With regard to ophthalmic surgery, collagen rich structures of the human eye continue to complicate surgical procedures despite recent improvements and advancements in the surgical field. In particular, a collagen rich structure, properly named the vitreous, continues to complicate many ophthalmic surgical procedures including, but not limited to, cataract surgery, retinal detachment and pars plana vitrectomy.
In cataract surgery, the collagen rich fibrils of the vitreous may protrude into the anterior chamber and cause irreversible corneal edema or retinal detachment. Following cataract surgery, the iridovitreal adhesions may block the posterior-anterior passage of aqueous humor. The blocked aqueous accumulates posteriorly and pushes the collagen fibrils of the vitreous into the anterior chamber. If these fibrils touch and remain in contact with the cornea, edema, corneal deterioration, striate keratopathy and bullous keratopathy could result. Current Concepts in Cataract Surgery, (ed). Jared Emery and David Paton, C. V. Mosby (publ.), 1974, pg. 329-335.
Other complications of cataract surgery, corneal edema or retinal detachment, occur when the collagen fibrils of the vitreous draw up and become incorporated into the wound.
There are many causes of retinal detachment. Basically, traction is exerted on the retina through the vitreous base attachment and this traction may cause the damage to the retina by tearing it or by detaching it from the global wall.
For example, in many persons between the ages of 50 and 80 years, syneresis, or vitreous liquification, predisposes a hole to develop in the retina at an area of vitreous traction. Another widespread problem is diabetic retinopathy which is observed in about 50 percent of the individuals who have had diabetes for 10 years, 75 percent of those who have had it for 15 years, and 95 percent of those who have had it for 25 years. Although not all suffer from visual impairment, the disease is the leading cause of blindness in the United States among persons between the ages of 20 and 65. Approximately 48,000 individuals in this country are legally blind as a result of it. Maugh, F. A., Science, 192:539 (1976).
Retinopathy results from the diabetes-induced deterioration of the retinal microvasculature. The cause of this deterioration is unknown. In the eye, small vessels become leaky and occluded and, occasionally, new vessels form on the surface of the retina. In the more severe form of the disease, known as proliferative retinopathy, new blood vessels protrude into the vitreous and eventually rupture and hemorrhage into the vitreous. Finally, fibrous scar tissue forms in association with the new vessels. This tissue may exert traction on the retina and detach it.
For many years, ophthamologists experienced many complications which resulted from surgical manipulation of the vitreous such as chronic inflammation, macular edema, corneal edema, retinal detachment, glaucoma and septic endophthalmitis.
The number of difficulties were significantly reduced in 1968 when Kasner and associates demonstrated that an eye can function after removal of the vitreous, as it can after lens removal. Kasner, D., Miller, G. R. and Taylor, W. H., Trans. Am. Acad. Ophthalmol. Otolarynogol., 72:410-418 (1968). The technique for vitreous removal was the traumatic open-sky technique, wherein the vitreous was removed through a large limbal incision.
The surgical trauma was significantly reduced with Machemer and associates' discovery in 1971. Machemer, R., Bueffner, H., and Norton, E. W., Trans. Am. Acad. Ophthalmol. Otolarngo., 75:813-820 (1971). They, Machemer, et al., discovered an instrument called the vitreous infusion suction cutter (VISC) which can be used for removal of the vitreous. Improvements to this instrument have been developed since that time; however, retinal tears are still reported as a frequent complication of pars plana vitrectomy. Retinal tears or retinal detachment usually occur because of traction of the retina at the vitreous base.
R. G. Michaels in Volume 80 at pages 24-29 of the American Journal of Ophthalmology (1975) reported, in 100 consecutive cases of citrectomy, a 19 percent incidence of anterior tears near the sclerotomy site, a 16 percent incidence of anterior tears at other sites of firm vitreoretinal adhesion, and a three percent incidence of dialysis (large retinal tear) in other quadrants.
Because a major portion of the ocular region contains collagenous material, the injecting and contacting of any portion of the ocular region with collagenase can be potentially hazardous to the continued enjoyment of vision by the mammal and, in particular, the human. However, this is not to suggest that injections into the ocular region have not been made. Indeed, U.S. Pat. No. 3,869,548 discloses a method of and a medicament for the treatment of ocular disorders such as diabetic retinopathy and degenerative maculation of the eye. The medicament used was a detoxified enzyme obtained from the venom of snakes.
Further, in an article entitled "The Effects of Bacterial Collagenase in Rabbit Vitreous" published in Volume 8 of the Canadian Journal of Ophthalmology, researchers O'Neill and Shea disclose the injection of collagenase into the rabbit vitreous for the purpose of studying the nature of vitreous fibrosis and its treatment. The investigators reported that the collagenase broke down the normal fibrillar structure of the vitreous but also broke down the internal limiting membrane and affected the inner layers of the retina.
In an apparent attempt to limit the deleterious effects of collagenase, the investigators suggested that larger doses of the enzyme could be used for shorter periods of time, between 24 and 72 hours, and that an intravitreal solution containing a collagenase inhibitor could be administered. Alternatively, the investigators suggested that the adverse effects of the collagenase could be managed by washing the enzyme out with a balanced salt solution in a manner similar to that used when chymotrypsin is utilized in chemical zonulysis. These suggestions are not viable solutions to the problem of collagenase destroying tissue which is not intended to be destoyed.